AN ACIDIC MOTIF WITHIN THE 3RD INTRACELLULAR LOOP OF THE ALPHA(2)C2 ADRENERGIC-RECEPTOR IS REQUIRED FOR AGONIST-PROMOTED PHOSPHORYLATION AND DESENSITIZATION

The alpha(2)C2 adrenergic receptor contains a highly acidic stretch of amino acids (EDEAEEEEEEEEEEEE) within the third intracellular loop. T o investigate the role of this region, we utilized site-directed mutag enesis to delete these 16 amino acids as well as to substitute them wi th glutamine, thereby conserving size but not charge. The wild-type an d mutated alpha(2)C2 receptors Were permanently expressed in CHO cells . Neither substitution nor deletion of this region affected receptor e xpression, agonist or antagonist binding affinities, guanine nucleotid e-sensitive formation of the high-affinity agonist-receptor-C protein complex, or functional coupling of the receptor to G(i). We considered that since alpha(2)C2 agonist-promoted desensitization is due to phos phorylation by the beta-adrenergic receptor kinase (or a related kinas e), that this region may be important for establishing the acidic mile au required by this kinase. Therefore, the consequences of 30 min of a gonist preexposure on subsequent alpha(2)C2-mediated inhibition of ade nylyl cyclase and on high-affinity agonist binding were determined for the wild-type and these two mutants. The wild-type alpha(2)C2 recepto r underwent similar to 52% functional desensitization and a similar to 40% loss of high-affinity binding after such exposure. In contrast, d eletion and substitution of this acidic stretch of amino acids ablated desensitization as assessed by both approaches. These results correla ted with those obtained in whole cell phosphorylation experiments. Cel ls expressing each receptor were incubated with [P-32]orthophosphate a nd exposed to agonist, and receptors were purified by immunoprecipitat ion. The deletion and the substitution mutant receptors underwent agon ist-promoted phosphorylation at levels of only 44 +/- 5% and 50 +/- 15 %, respectively, relative to wild-type alpha(2)C2. Therefore, this uni que acidic motif in the alpha(2)C2 receptor is necessary for full phos phorylation induced by agonist which, in turn, is required for agonist -promoted desensitization.